Extensive study of HIV+ individuals in recent years has brought to light many examples of antibodies which can neutralize a broad range of HIV strains and protect against viral challenge in animal models of infection. One particularly promising antibody family, PGT121-123, has been shown to bind to an epitope involving a combination of V3 and V1 peptide together with several glycans, possibly of both high mannose and complex type. The goal of this project is to develop immunogens which mimic these glycopeptide structures, and test their ability to elicit antibodies with broadly-neutralizing, PGT122-like specificity. Because the glycan and peptide components of the PGT122 epitope are discontinuous within the gp120 primary sequence, it is quite challenging to design a single stretch of glycopeptide which could reconstitute all of the epitope elements in their native 3D orientation. To address this challenge, our group has developed a way to design glycopeptide epitope mimics by directed evolution. In Aim 1, we will adapt this method to the evolution of glycopeptides containing two different glycans, which will be useful for incorporation of both complex- and high mannose glycans into our glycopeptide libraries. In Aim 2, we will generate a library of ~10^13 random glycopeptides, then select and amplify those which bind best to PGT122. After multiple rounds of selection/amplification we will obtain glycopeptides which are very tightly recognized by PGT122. We will also do an analogous selection to obtain glycopeptides/peptides which are recognized by germline (gl) PGT122, and all promising constructs will be biophysically and structurally characterized. In Aim 3, we will then test the immunogenicity of PGT122 epitope mimics in rabbits. To address the possibility that bnAbs may only arise by evolving from the correct precursor germline antibodies, we will also conduct immunogenicity studies with the germline-targeted immunogens. In collaboration with David Nemazee at Scripps, we will investigate the ability of these constructs to activate gl-PGT122 B cells and stimulate a gl-PGT122 antibody response in knock-in mice.